Detection of non-naturally occurring nucleotide sequence mutations has been approached by performing studies on cells in culture or on live animals based on alterations in phenotype. Tests on cells in culture using bacterial or animal cells or cell lines permits the rapid screening of a large number of cells for the appearance of an altered phenotype. The appearance of an altered phenotypic trait reflects the occurrence of a mutation in the test gene.
Previous attempts to identify genetic mutations have involved genetic mutation analysis based on phenotypic screening (Russell et al., 1979, Proc. Nat. Aca. Sci. USA 76:5818; Russell et al., 1982, Proc. Nat. Aca. Sci. USA 79:3589). That is, a phenotypic abnormality, such as alteration from wild-type (e.g. coat color in mice), is detected in the F1 offspring of a mutated animal, or in subsequent generations. Thus, Russell et al. assess mutation frequencies in a number of loci by identifying a mutant phenotype and correlating phenotype with a mutation at a corresponding locus. This is known as the `specific-locus method` of calculating the frequency of mutations in a given locus. However, observation of a mutant phenotype does not directly identify the gene which is mutated, although for phenotypes known to be the result of mutation of a particular gene, it may be inferred and subsequently tested. Phenotypes of interest can serve as a guide to study particular genes, using conventional mapping and positional cloning techniques to identify a gene or genes relating to the phenotype. This approach relies on the occurrence of a phenotype which is used to score for a mutation, and the phenotype acts as a guide to the mutated gene.
Johnson et al., (1981, Proc. Nat. Aca. Sci. USA 82:5829) and Lewis et al., (1985, Proc. Nat. Aca. Sci. USA 82:5829) disclose a protein phenotype screen which detects electrophoretic mobility changes in proteins to test for induced genetic mutations. Protein extracts are isolated from a number of mutagenized animals, and specific proteins are assayed to look for abnormal electrophoretic migrations. This system identifies a change in phenotype in a protein in order to find a mutation in its corresponding gene.
The disadvantage of phenotypic screening for gene mutations is that the analysis of mutation distribution is always based on the window of observation that is permitted by the selective mutation system used, in which an alteration in a cell phenotype indicates that a mutation has occurred in a particular gene. The chief drawback of mutation assays involving phenotypic selection is that mutation analysis is confined to those genetic alterations which produce an altered gene product which is detectable via a phenotypic screen. Therefore, a phenotype must be matched with the mutation prior to detection or characterization of the mutant gene itself. For example, U.S. Pat. No. 5,347,075 discloses mutagenesis testing using a transgenic animal carrying a lacZ reporter test gene, wherein either cells containing the test gene or the animal itself is mutated, the `mutated` test gene is cloned in bacteria and then grown on X-gal indicator plates. Mutations in the reporter test gene are thus indicated phenotypically as white plaques rather than blue plaques.
Previous attempts to identify genetic mutations have also involved purely genotypic mutation analysis in vitro, or non-phenotypic selection of mutations (Palombo et al., 1992, Nucl. Acids Res. 20:1349; Chiocca et al., 1992, Proc. Nat. Aca. Sci. USA 78:3138). In these analyses, cells in culture are mutagenized, the DNA isolated, and tests are performed to detect mutations, for example, via changes in specific restriction endonuclease sites (RFLP analysis). Although this procedure tests DNA directly for induced mutations, it has been adapted solely for mutagenesis of cultured cells.
U.S. Pat. No. 5,045,450 discloses a method of determining a mutation spectrum in a DNA sequence of interest that is present in a population of cells. The method includes detecting spontaneous mutations in a DNA sample wherein DNA is extracted from the tissue to be analyzed, hybridized to form duplexes with nonmutated DNA, and subject to DNA gradient gel electrophoresis to detect single base changes.
Mutation detection can be divided into two categories: the detection of mutations in candidate disease genes; and the identification of mutations in known disease genes, each of which have different requirements. The detection of mutations in candidate disease genes has been based on the mapping of a particular phenotype to a particular chromosome region and the examination of all genes mapping to this region for mutations in order to identify the gene responsible for the disease.
Animal models of disease have been produced in the prior art via phenotypic observation of a mutated animal. See, for example, Harding et al., 1992, Proc. Nat. Aca. Sci. USA 89:2644, in which a mouse mutant with sarcosinemia was found by screening the progeny of ENU-mutagenized mice for aminoacidurias; and Bode et al, 1988, Genetics 118:299, in which ENU-mutagenesis was used to screen for defects in phenylalanine metabolism by detecting elevated serum levels of phenylalanine. Mouse models of disease have also been produced via targeted mutagenesis involving targeting of a specific gene in ES cells, production of a mouse from the mutated ES cells, and ascertainment of phenotype. In such targeted mutations, the mutated gene is typically a "knockout", i.e., in which a mutation is generated which fully or partially inactivates the gene. For example, see Sadlack et al., 1993, Cell 75:253, in which mice deficient for IL-2 were constructed; and Colledge et al., 1995, Nat. Genet. 10:445, in which mice were generated carrying a mutation in the cystic fibrosis gene.
One object of the invention is to provide mutational screening methods based on genomic and genetic techniques, rather than on phenotypic observation, to identify and characterize a mutation in a gene of interest.
Another object of the invention is to identify and characterize genes via mutagenesis in order to identify genes encoding products which may have therapeutic benefit.
Another object of the invention to provide methods for identifying mutations in a gene of interest which do not rely solely upon prior matching of a gene with a disease.
Another object of the invention is to provide methods for identifying mutations in a gene of interest which do not rely upon prior matching of a phenotypic mutation to a gene.
There is a need in the art for a direct test for mutations in the DNA of animals without using a phenotypic guide.